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Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

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PfA-M1 [1-30]-GFP yields a 32-kDa protein strictly associated with the parasite membranes that localizes to the ER. A. Soluble proteins (lanes 2 and 3) and membrane proteins (lanes 4 and 5) from parasites expressing PfA-M1-GFP chimera (51, lanes 2 and 4) or PfA-M1 [1-30]-GFP chimera (lanes 3 and 5) were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-GFP antibodies as described in the Methods section. PfA-M1-GFP chimera was detected at ~150-kDa in soluble (lane 2) and membrane (lane 4) fractions of the parasites while the ~32-kDa PfA-M1 [1-30]-GFP fusion protein is strictly insoluble (lane 5). Note that free soluble GFP is produced from PfA-M1-GFP chimera (lane 2), but not from PfA-M1 [1-30]-GFP chimera (lane 3). Mw markers in kDa are in lane 1. B. Live imaging of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera. Top row: ring stages; bottom row: young schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm. C. Immuno-fluorescence analysis of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera, fixed by using 3.7% formaldehyde and analysed as previously described [4,6] by using rabbit anti-PfBip [21](red) and mouse anti-GFP (green) antibodies. Top row: trophozoite stages; bottom row: schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm.
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Figure 4: PfA-M1 [1-30]-GFP yields a 32-kDa protein strictly associated with the parasite membranes that localizes to the ER. A. Soluble proteins (lanes 2 and 3) and membrane proteins (lanes 4 and 5) from parasites expressing PfA-M1-GFP chimera (51, lanes 2 and 4) or PfA-M1 [1-30]-GFP chimera (lanes 3 and 5) were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-GFP antibodies as described in the Methods section. PfA-M1-GFP chimera was detected at ~150-kDa in soluble (lane 2) and membrane (lane 4) fractions of the parasites while the ~32-kDa PfA-M1 [1-30]-GFP fusion protein is strictly insoluble (lane 5). Note that free soluble GFP is produced from PfA-M1-GFP chimera (lane 2), but not from PfA-M1 [1-30]-GFP chimera (lane 3). Mw markers in kDa are in lane 1. B. Live imaging of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera. Top row: ring stages; bottom row: young schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm. C. Immuno-fluorescence analysis of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera, fixed by using 3.7% formaldehyde and analysed as previously described [4,6] by using rabbit anti-PfBip [21](red) and mouse anti-GFP (green) antibodies. Top row: trophozoite stages; bottom row: schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm.

Mentions: In order to test whether the N-terminal segment of PfA-M1 behaves as a signal peptide and has the capacity to target proteins to the ER, this region was fused to a reporter GFP protein using the Multisite Gateway™ technology system developed by Van Dooren et al [27] for Plasmodium. Transfectant parasites express the expected ~32-kDa fusion protein that is entirely associated with the parasite membrane fraction (Figure 4A). Interestingly, no soluble GFP was cleaved off this construct (Figure 4A). Live imaging on parasites transfected with this PfA-M1 [1-30]-GFP construct revealed a pattern characteristic of ER labelling [27,33] (Figure 4B) that was confirmed by immuno-fluorescence (Figure 4C). These results strongly suggest that the N-terminal segment of PfA-M1 has the capacity to target the protein to the ER of the parasite. But in the absence of the downstream PfA-M1 sequence, this protein remains blocked in the ER and attached to the membrane.


Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

PfA-M1 [1-30]-GFP yields a 32-kDa protein strictly associated with the parasite membranes that localizes to the ER. A. Soluble proteins (lanes 2 and 3) and membrane proteins (lanes 4 and 5) from parasites expressing PfA-M1-GFP chimera (51, lanes 2 and 4) or PfA-M1 [1-30]-GFP chimera (lanes 3 and 5) were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-GFP antibodies as described in the Methods section. PfA-M1-GFP chimera was detected at ~150-kDa in soluble (lane 2) and membrane (lane 4) fractions of the parasites while the ~32-kDa PfA-M1 [1-30]-GFP fusion protein is strictly insoluble (lane 5). Note that free soluble GFP is produced from PfA-M1-GFP chimera (lane 2), but not from PfA-M1 [1-30]-GFP chimera (lane 3). Mw markers in kDa are in lane 1. B. Live imaging of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera. Top row: ring stages; bottom row: young schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm. C. Immuno-fluorescence analysis of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera, fixed by using 3.7% formaldehyde and analysed as previously described [4,6] by using rabbit anti-PfBip [21](red) and mouse anti-GFP (green) antibodies. Top row: trophozoite stages; bottom row: schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2914058&req=5

Figure 4: PfA-M1 [1-30]-GFP yields a 32-kDa protein strictly associated with the parasite membranes that localizes to the ER. A. Soluble proteins (lanes 2 and 3) and membrane proteins (lanes 4 and 5) from parasites expressing PfA-M1-GFP chimera (51, lanes 2 and 4) or PfA-M1 [1-30]-GFP chimera (lanes 3 and 5) were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-GFP antibodies as described in the Methods section. PfA-M1-GFP chimera was detected at ~150-kDa in soluble (lane 2) and membrane (lane 4) fractions of the parasites while the ~32-kDa PfA-M1 [1-30]-GFP fusion protein is strictly insoluble (lane 5). Note that free soluble GFP is produced from PfA-M1-GFP chimera (lane 2), but not from PfA-M1 [1-30]-GFP chimera (lane 3). Mw markers in kDa are in lane 1. B. Live imaging of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera. Top row: ring stages; bottom row: young schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm. C. Immuno-fluorescence analysis of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera, fixed by using 3.7% formaldehyde and analysed as previously described [4,6] by using rabbit anti-PfBip [21](red) and mouse anti-GFP (green) antibodies. Top row: trophozoite stages; bottom row: schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml-1. cc stands for "colour combine". Scale bar, 5 μm.
Mentions: In order to test whether the N-terminal segment of PfA-M1 behaves as a signal peptide and has the capacity to target proteins to the ER, this region was fused to a reporter GFP protein using the Multisite Gateway™ technology system developed by Van Dooren et al [27] for Plasmodium. Transfectant parasites express the expected ~32-kDa fusion protein that is entirely associated with the parasite membrane fraction (Figure 4A). Interestingly, no soluble GFP was cleaved off this construct (Figure 4A). Live imaging on parasites transfected with this PfA-M1 [1-30]-GFP construct revealed a pattern characteristic of ER labelling [27,33] (Figure 4B) that was confirmed by immuno-fluorescence (Figure 4C). These results strongly suggest that the N-terminal segment of PfA-M1 has the capacity to target the protein to the ER of the parasite. But in the absence of the downstream PfA-M1 sequence, this protein remains blocked in the ER and attached to the membrane.

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Show MeSH
Related in: MedlinePlus